Maltosylisothiocyanate (MITC) has been synthesized and found to be a specific covalent affinity label for the glucose transporter of human erythrocyte membrane. The transporter has been identified as a 100,000 dalton component of Band 3 which can be converted to a Band 4.5 component by endogenous or exogenous proteolysis. I propose to: 1) Localize the site of MITC reaction with the transporter with respect to its topography in the membrane, its position in the primary structure of the protein, the site of cytochalasin B interaction, and the site of fluorodinitrobenzene reaction. 2) Compare its site of reaction with that of affinity labels for the anion transporter, which is also present in Band 3. 3) Utilize appropriately designed affinity labels in conjunction with other techniques to test the hypothesis that the transporter forms a channel which has different conformations in the "in" and "out" states. 4) Determine whether MITC serves as an affinity label for the glucose transporter of the rat adipocyte. 5) Determine whether insulin treatment of adipocytes results in increased specific incorporation of (14C)-MITC into their glucose transporter, whether this can be correlated with insulin enhancement of glucose transport, and whether cytochalasin B antagonizes this action of MITC. 6) Acertain whether the diminshed rate of glucose transport in adult nonprimate erythrocytes is correlated with decreased numbers of Band 3 molecules which are reactive with MITC and other affinity labels or whether it results from some other alteration in their membranes. The effect of erythrocyte age on the rate of glucose transport and MITC incorporation will also be measured in both nonprimate and human erythrocytes.